Endostatin protein is a potent and specific inhibitor of endothelial proliferation and angiogenesis. Systemic or localized therapy with endostatin protein causes a nearly complete suppression of tumor-induced angiogenesis, and it exhibits strong anti-tumor activity. Endostatin protein has been isolated and purified from the murine hemangioendothelioma cell line EOMA, and is capable of inhibiting endothelial cell proliferation in cultured endothelial cells. Endostatin protein is a protein with a molecular weight of approximately 18,000 to 20,000 Daltons (18 to 20 kDa).
Endostatin protein is useful for treating diseases and processes mediated by undesired and uncontrolled angiogenesis by administering to a human or animal with the undesired angiogenesis a composition comprising a substantially purified endostatin protein or endostatin protein derivative, in a pharmaceutically acceptable carrier, in a dosage sufficient to inhibit angiogenesis. Endostatin protein is particularly useful for treating or for repressing the growth of tumors. Administration of endostatin protein to a human or animal with prevascularized metastasized tumors prevents the growth or expansion of those tumors.
Isolation and purification of proteins, such as endostatin protein, in high yield from biological material, such as tissue extracts, cell extracts, broth from incubation systems, and culture medium is often frought with problems in view of the numerous proteins and other undesirable molecules present in an homogenate or extract. What is needed are recombinant methods of producing endostatin protein that will provide the large amounts of endostatin protein required for clinical use, including, but not limited to, cancer therapy. Such methods should produce endostatin protein in an efficient and convenient manner in a culture broth which is amenable to procedures designed to recover and purify endostatin protein in high yields. Separating a specific protein of interest from potential contaminants presents a challenge in view of numerous factors, such as contamination of cellular homogenates with proteolytic enzymes that may digest the protein. Other undesirable cellular constituents that may be present in homogenates, include but not limited to, pigments, cytochromes, lipids, free radicals, oxidases and other lysosomal enzymes, and oxides. Some of these substances may affect the protein of interest by stripping electrons, affecting disulfide bonds and changing the conformation of the protein.
Centrifugation of cells, including yeast, bacteria, insect and other cells used for recombinant production of proteins, such as endostatin protein, may result in damage to the cells with concomitant release of undesirable biological material. What is needed is a method for recovery and purification of protein, such as endostatin protein, which does not employ centrifugation.
Methods for recombinant production, recovery and purification of endostatin protein on a large scale are required to produce and isolate the amounts of purified endostatin protein needed for administration to patients and also for research purposes.
Solutions are also needed for storage of endostatin protein after recovery and purification, which provide desired solubility and stability of endostatin protein. Since large quantities of purified endostatin protein are needed for use in the clinic and in research laboratories, it is necessary to determine the proper conditions for solubility of the endostatin protein so that it may be stored conveniently at different temperatures while preserving biological activity. Optimal buffer systems are also needed for lyophilization of endostatin protein. Lyophilization of endostatin protein would permit periods of extended storage before conveniently reconstituting endostatin protein in an appropriate buffer system, as needed, before use. It is advantageous to reconstitute lyophilized proteins prior to clinical administration to humans or animals. Accordingly, what is needed are optimal solubility conditions for the endostatin protein, especially recombinantly produced endostatin protein. What is also needed are methods for recovering and/or purifying endostatin protein, particularly recombinantly-produced endostatin protein. Such conditions involving the optimal solutions and solubility conditions to facilitate storage, lyophilization and reconstitution, must not have deleterious effects on the biological activity of endostatin protein.
Also needed is a method for purifying recombinantly-produced endostatine protein which avoids the need for centrifugation of the culture broth, thereby avoiding problems associated with cell lysis. This method should be capable of use on a large scale to recover and purify endostatin protein in quantities needed for clinical administration and research.
What is also needed is a method for purifying recombinantly-produced endostatinprotein which minimizes contamination with cytochromes, pigments, enzymes, and other undesirable cellular constituents.
Also needed are solutions for storage of endostatin protein following the recovery and purification process which optimizes solubility properties of endostatin protein.